FINAL REPORT

The exchange of the technologies between the two laboratories was performed. The ASU group is a leader in the development of the sample preparation techniques for various scanning probe microscopy applications. One of these techniques was successfully implemented at MSU for two projects. In one the conformational study of viral RNAs was performed. In these experiments we used as a substrate the mica surface, chemically modified by the method developed at Y. Lyubchenko laboratory (ASU). Movement protein (MP) molecules were found to be distributed along the chain of RNA and the structure of MP–RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60–100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive 'beads-on-a-string' into a 'thick string' form that was partly resistant to Rnase. The role of hydrodynamic flow and Coulomb interaction, responsible for the conformation of the absorbed RNA molecules on chemically modified surface of mica, was estimated The results were published in [4]. The study of bacterial cell was done in the second project [1,5,6]. We visualized bacterial cells of different taxonomy groups. Special emphasis was done on the study of mechanical properties of the bacterial membranes in liquid environments and in air. Noticeable difference in the mechanical rigidity of Arthrobacter globoformis bacteria in vegetative and mummy forms was revealed. SPM experiments for the visualization of Escherichia coli bacteria degradation induced by lysozyme protein (formation of spheroplasts) were performed [2]. The results of this project were presented the EMBL Conference "Scanning Probe Microscopy, Cantilever Sensors and Nanostructures and summarized in the joint paper [2].       Both groups were actively participated in development of novel technique for the tip and the substrates functionalization. In one paper (ref. [7]) a procedure for direct functionalization of silicone nitride AFM probes with 3-aminopropyltriethoxysialne (APTES) was described. The probes were modified in vapors of APTES at ambient conditions (AP-tips). The functionalization of tips was monitored by fluorescent microscopy and the force measurements with AFM. AP-tips were derivatized with a non-fluorescent quinoline analogue (FQ) which reacts with primary amines to form a strongly fluorescent isoindole. AP-tip fluorescence was significantly higher than control probes. Amino modified tips manifest a strong adhesion to freshly cleaved mica and this behavior is qualitatively similar to the interaction of unmodified silicone nitride probes with AP-mica. The adhesion of AP-tips to mica or AP-mica to unmodified tips decreases as ionic strength increases indicating a dominant electrostatic component of the adhesio effect. This interpretation is supported by the experiments at different pH and the reaction of AP-tips with glutaraldehyde. The adhesion effect is very small at basic pH due to the deprotonation of amino groups. The reaction of immobilized amines by the treatment of the tips with glutaraldehyde decreases the tip-surface interaction dramatically. Like AP-mica, AP-tips stable during storage and thus can be used for additional derivatization steps. (a copy is appended). The second paper (ref. [8]) describes a novel procedure of surface functionalization based on the use of 1-(3-Aminopropyl)silatrane (APS) instead of our early procedure utilizing aminopropyl triethoxy silane (APTES). Unlike APTES, APS is less reactive and extremely resistant to hydrolysis and polymerization at neutral pH. The kinetics of DNA adsorption to APS-mica was studied. The results are consistent with a diffusion controlled mechanism suggesting that DNA molecules equilibrate onto the surface upon immobilization. This conclusion is supported by the data on imaging of supercoiled DNA, the labile conformations of which are very sensitive to the conditions at the surface-liquid interface. In addition, we demonstrated directly that DNA molecules move along the surface if the sample is imaged in aqueous solution without drying of the sample. Using the time-lapse mode of AFM imaging we visualized an intramolecular conformational transition in supercoiled DNA. These data show directly that changes in local DNA conformations are linked with global reorganization of the DNA molecule.       One of the major efforts of the MSU group is focused on the software development for image analysis applicable primarily to AFM images. This group has has developed one of most versatile software packages FemtoScan. During the grant period this software was dramatically improved. The development of novel features was stimulated by the work on joint projects during the visits of the MSU researchers at ASU. Novel version Femtoscan Online features a number of novel options allowing the SPM image processing that were utilized for topographical analysis of various biological samples. The Femptoscan Online software is routinely used at ASU during the AFM data analysis of DNA, protein-DNA complexes and complex self-assembled protein aggregates.      In summary, this NATO award helped us establish a very productive collaboration. It allowed both groups to identify the common interests, to substantially improve available techniques, to implement novel experimental and computer analysis approaches, and extend the areas of research programs. Importantly, the current progress made a firm foundation for further extension of this fruitful collaboration towards dynamic studies bimolecular complexes that we plan to pursue via the second LST request.

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